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22 views

cbc-procedure

Uploaded by

drbouchr1
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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WBC COUNT

TLC or WBC pipette:


This pipette (also called Thoma pipette) long stem is divided into two parts:
1.The long stem is marked with 0.5 and 1.0
2.While the short arm after the bulb is marked 11.
3.Its central portion is a bulb or a globular shape with one white bead in it.
4.Rubber tubing is attached to suck the blood.
5.Ultimately the dilution of the blood to the TLC fluid is 1:20

WBC pipette

WBC pipette method:


1 .Fill the blood into the 0.5 marks and then add the TLC solution.
2.Fill the pipette with the TLC solution to point 11. Avoid bubbles inside.
3.Remove the rubber tubing.
4.Seal both ends or hold in between two fingers.
5.or can put this pipette on the mechanical device to shake it.
6.Shake for 1 minute or preferably for 2 minutes.
7.Shaking is important before filling the Neubauer chamber.
8.After thorough mixing, discard the first few drops (3-5) and then gently fill the chamber until the
platform is filled.
8.The capillary action will draw the fluid.
9.Allow the chamber on the microscope stage for 2 to 3 minutes till the cells are settled.
Neubauer chamber counting of white blood cell (TLC)
Procedure to fill the Neubauer chamber
WBCs counted in one of the large square as a sample
1.Count the cells in the Neubauer chamber. These are counted in the four large corner squares labeled
as WBC and if the number is Y.
2.One large area is 1 x 1 mm, and the depth is 0.1 mm.
3.Total area counted in 4 large squares = 4 x 1 x o.1 = 0.4 µL (4/10).
4.Y x 10/4 is the total WBC in the cell in 1 µL.
5.Now dilution is 1:20.
6.Number of WBC in 1µL = Y x 10 x 20/4 = Y x 50 = Total WBC count.
7.Total TLC = counted cells (Y) x 50 = TLC/cmm.

Normal values of total leucocytes are:

Source 2
•Adult /child = 5000 to 10,000 /cmm
•Child ≤2 years = 6200 to 17000 /cmm.
•Newborn = 9000 to 30,000 /cmm

Other sources
1.At birth = 10,000 to 25,000/cmm
2.Infants = 8000 to 15,000/cmm
3.Adults = 4000 to 10,000/cmm
4.Pregnant ladies = 12,000 to 15,000/cmm
Calculations:

1. The calculation formula for hemocytometer cell counts determines the number of cells within 1 µL (1
mm3) of blood. To make this determination, the total number of cells counted must be corrected for the
initial dilution of blood and the volume of diluted blood used.

The standard dilution of blood for leukocyte counts is 1:20; therefore the dilution factor is 20. The
volume of diluted blood used is based on the area and depth of the counting area. The area counted is 4
mm2 and the depth is 0.1 mm; therefore the volume factor is 0.4 mm3. Total number of cells counted •
dilution factor • 1/volume factor = cells/mm3.

For example if 150 cells were counted in the four corner squares the WBC count is:

150 × 20 × 1/0.4 = 7,500 cells/mm3 or 7.5 × 109 /L

RBC COUNT

RBC Pipette
It is commonly used to dilute the blood sample with the RBC diluting fluid. It gives a dilution of 1:100
and 1:200. The reading starts from 0.5 to the endpoint of 101. There is a red bead within the RBC
pipette, which mixes the RBC specimen with the diluting fluid.
There is a mouthpiece attached towards the end of the suction rubber tube. Through the mouthpiece,
the blood is sucked upto a point 0.5 and diluting fluid upto the endpoint 101. The diagram below
provides a well-labelled presentation of an RBC pipette.
Procedure:
Take the blood sample upto a point (0.5). Then, wipe the RBC pipette’s tip using blotting paper.
After that, suck RBC diluting fluid or diluent upto a mark 101.
Horizontally rotate the RBC pipette by using your palms.
Loading the sample over the haemocytometer slide:

Take a clean, grease-free haemocytometer slide and cover glass.


Place the cover glass on top of the haemocytometer’s lined region.
Before loading the RBC sample into the haemocytometer, discard 1-2 drops.
Then, you should carry the RBC pipette at an angle (45 degrees) and load a small volume of RBC sample
towards the edge of a cover glass.
Wait for 3-5 minutes in order to settle down the RBCs in the chamber.
Observe the prepared slide under the microscope to count the number of RBCs manually.

RBC Count under Microscope

First, focus the rulings of the haemocytometer slide using a 10X objective lens. Using coarse and fine
adjustment knobs, focus on the five squares of the large central square to count the number of red
blood cells under the 40X objective.
A diagram below represents the pattern to count RBCs in all the five medium squares of a large central
square. As already discussed, each medium square possesses 16 small squares. We need to manually
count the number of RBCs in five medium squares via hand tally.
In each square, you need to count the red blood cells located within the square. The red lines in the
upper and right corners indicate the areas not to count RBCs, whereas green lines indicate the areas to
count the RBCs.

Calculation of RBCs

Total RBCs/µL = Number of RBCs counted X Dilution factor / Area X Depth

 The number of red blood cells (N) =?


 Dilution factor = 1:200 or 200
 A large central square is subdivided into 25 medium squares or sub squares. So, the area will be one
sq. mm. The number of RBCs is enumerated in 5 squares out of 25 squares. So, the area of 5 small
squares will be 5/25 or 1/5 sq. mm.
 Depth of the sample = 0.1 mm
Total RBCs = N X 200 / 1/5 X 0.1 = N X 200 X 50 = N X 10,000 cells/µL
Suppose, N or number of RBCs in the five squares is 486, then the equation will be represented as:

Total RBCs = 486 X 10,000 = 48, 600, 000 cells/µL

A normal RBC count would be: men – 4.7 to 6.1 million cells per microlitre (cells/mcL) women – 4.2 to
5.4 million cells/mcL.

PLATELET COUNT
Procedure:
1. Use the RBC pipet and use the platelet diluting fluid.
2. Charge the counting chamber and stan for 10inutes in a wet chamber.
3. Count all the 25 squares in the central square of the counting chamber
4. No. of platelets counted X 1,000_ total number of platelets

DIFFERENTIAL COUNTING

1. Prepare a blood smear


2. Let it dry and stain with Giemsa stain or any hematology stain.
3. Air dry and observe under the OIO.

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